Secretory production of cell wall components by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> protoplasts in static liquid culture

When protoplasts of Saccharomyces cerevisiae T7 and IFO 0309 are cultured in a static liquid culture at 2.5 × 10⁶ protoplasts/ml, cell wall regeneration does not occur and cell wall components (CWC) are released into the culture broth. By using a specialized fluorometer, the concentrations of CWC could be measured on the basis of the fluorescence intensity of the CWC after staining with Fluostain I. The inoculum concentration, pH, and osmotic pressure of the medium were important factors for the production of CWC in culture. Under optimal culture conditions, S. cerevisiae T7 protoplasts produced 0.91 mg/ml CWC after 24 h. The CWC induced the tumor necrosis factor-α production about 1.3 times higher than that of the commercially available β-1,3/1,6-glucan from baker’s yeast cells.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Papulacandin B: Inhibitor of biogenesis of (1→3)-β-d-glucan fibrillar component of the cell wall ofSaccharomyces cerevisiae protoplasts

The effect of papulacandin B on regenerating protoplasts ofSaccharomyces cerevisiae was studied by light and electron microscopy. In liquid media it inhibited the biogenesis of (1→3)-β-d-glucan fibrillar nets; as a result, the protoplasts did not grow polarly but only spherically. The effect was reversible. Instead of the nets the inhibited protoplasts synthesized only individual microfibrils soluble in hydroxide; these were not joined in the nets and were partially masked by amorphous material. The microfibrils disintegrated after lysis and did not maintain the shape of protoplasts. Protoplasts inhibited in solid media grew spherically up to 25 μm but they did not divide or revert or revert, in spite of forming cell walls. These walls were amorphous and fragile and they disintegrated during preparation. Papulacandin B did not decrease the viability of protoplasts and did not interfere with their growth, biogenesis of alkali-soluble glucan microfibrils or amorphous wall matrix. It inhibited specifically the synthesis of alkali-insoluble branched (1→3)-β-d-glucan, a necessary building unit required for the formation of the fibrillar component of the cell wall responsible for the cell wall shape, its rigidity and tensile strength.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Solid state bioreactor production of transglutaminase by Amazonian Bacillus circulans BL32 strain

In this work, we investigated the production of transglutaminase (TGase) by an Amazonian isolated strain of Bacillus circulans by solid-state cultivation (SSC). Several agro-industrial residues, such as untreated corn grits, milled brewers rice, industrial fibrous soy residue, soy hull, and malt bagasse, were used as substrates for microbial growth and enzyme production. Growth on industrial fibrous soy residue, which is rich in protein and hemicellulose, produced the highest TGase activity (0.74 U g⁻¹ of dried substrate after 48 h of incubation). A 2³ central composite design was applied to determine the optimal conditions of aeration, cultivation temperature and inoculum cell concentration to TGase production. The best culture conditions were determined as being 0.6 L air min⁻¹, 33 °C and 10 log ₁₀ CFU g⁻¹ of dried substrate, respectively. Under the proposed optimized conditions, the model predicted an enzyme production of 1.16 U g⁻¹ of dried substrate, closely matching the experimental activity of 1.25 U g⁻¹. Results presented in this work point to the use of this newly isolated B. circulans strain as a potential alternative of microbial source for TGase production by SSC, using inexpensive culture media.     

Production and regeneration of protoplasts from <Emphasis Type="Italic">Grateloupia turuturu</Emphasis> Yamada (Rhodophyta)

Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 10⁷ protoplasts g^-1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 10⁶ cells mL^-1 in culture medium and developed into microthalli of a line of two to six cells.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.     

Changes in cell wall composition of deformedras1 − cells ofSchizosaccharomyces pombe

Disruption of theSchizosaccharomyces pombe ras1 gene results in a morphological transformation to large spheres, in contrast to wild-type cells which grow as rods. Chemical analysis of isolated cell walls showed no significant changes in saccharide content but an increase in protein and phosphate contents inras1 ⁻ walls relative to parent walls. Polymers tightly bound to the cell wall were solubilized by SDS treatment. Several compounds with molar mass ranging from 22 to 130 kDa and more were resolved by gel filtration and SDS-PAGE. Among low-molar-mass species, a component moving as a band at 31 kDa was conspicuous inras1 ⁻ cell walls. It was solubilized by heating in Tris-HCl buffer and shown to have a β-1,3-glucanase activity against laminarin. The level of the enzyme was by 30% higher in theras1 ⁻ cell wall than in the wild-type cell wall. This enzyme may participate in the remodelling of the rigid glucan network and account (at least partially) for the aberrant cell shape. Theras1 ⁻ cell wall contained a high level of charged polymers, especially phosphoproteins, raising the appealing possibility thatras1 ⁻ is involved in a putative kinase cascade required to sense and respond to external stimuli destined for the cell wall. Although the present study shows thatras1 loss of function and altered cell wall composition are closely linked defects, it has still to be shown that theras1 protein is directly involved in alterations found in the mutant cell walls.     

Chitinase Production in Solid-State Fermentation from <Emphasis Type="Italic">Oerskovia xanthineolytica</Emphasis> NCIM 2839 and its Application in Fungal Protoplasts Formation

The present study reports the economic production of thermostable chitinase production from Oerskovia xanthineolytica NCIM 2839 by solid-state fermentation (SSF) technique and its application in fungal protoplasts formation. The Oerskovia xanthineolytica NCIM 2839 was found to produce thermostable chitinase 148 U g⁻¹ of solid substrate in SSF using wheat bran with colloidal chitin as base. Protoplasts of A. niger were formed by using crude chitinase produced in SSF and formed protoplasts were confirmed by using scanning electron microscopy. This is the simple and economical method for protoplast formation which makes it possible applications in strain improvement of various fungi by protoplasts fusion in Biotechnological industries.     

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

Efficient plant regeneration from protoplasts of <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis> via organogenesis

A simple and efficient protocol for plant regeneration from protoplasts of the potted plant Kalanchoe blossfeldiana Poelln. is reported. Mesophyll protoplasts were isolated from axenic leaves after a preculture. The enzymatic digestion of the tissue with a solution containing 0.4% Cellulase Onozuka R-10 and 0.2% Driselase yielded 6.0 × 10⁵ protoplasts per gram fresh weight after density gradient purification. Protoplasts were cultured in the dark at an initial density of 1 × 10⁵ protoplasts per milliliter in a liquid medium with 320 mM mannitol, 130 mM sucrose, 2.3 μM 2,4-dichlorophenoxy acetic acid (2,4-D), 5.4 μM 1-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzyladenine (BA). Cell wall regeneration was observed within 4 days of culture and cell division began after 5–7 days. When cultured in a liquid medium with 5.4 μM NAA and 8.9 μM BA, protoplast-derived colonies proliferated until small visible calli, and adventitious buds appeared after transfer to photoperiod conditions. Developed shoots were rooted on a solid medium supplemented with 0.6 μM indole-3-acetic acid (IAA) and successfully established under greenhouse conditions. The process required 4 months from isolation to rooted plants and the best conditions found gave a plant regeneration efficiency of 6.4 plants per 1 × 10⁵ protoplasts. This is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species.     

Plant regeneration from protoplasts of <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA) via somatic embryogenesis

A protocol for plant regeneration from protoplasts of Musa acuminata cv. Mas (AA) via somatic embryogenesis was developed. Viable protoplasts were isolated from embryogenic cell suspensions at a yield of 1.2 × 10⁷ protoplasts/ml packed cell volume (PCV). Liquid and feeder layer culture systems with medium-A and medium-B were used for protoplast culture. In liquid culture system, medium-B was more efficient for inducing cell division (17.5% at 14 days) and colony formation (6.7% at 28 days) than medium-A. However, all protoplast-derived cell colonies (PDCC) obtained from liquid culture system could not develop further. In feeder layer culture system, there was no significant difference between medium-A and medium-B on cell division and colony formation of the cultured protoplasts, and the cell division frequency at 14 days and colony formation frequency at 28 days were 24.5% and 11.2%, respectively, in medium-B. Comparative study on the effects of BAP (2.2 μM, 4.4 μM, 8.8 μM), zeatin (0.4 μM, 0.8 μM, 1.2 μM) and TDZ (0.2 μM, 0.4 μM, 0.6 μM) on embryo formation of PDCC from feeder-layer culture indicated that TDZ was best. TDZ at 0.4 μM induced 7906 mature embryos per ml PCV PDCC, which was 4-fold the frequency as with BAP at 4.4 μM, 7.5-fold as with zeatin at 0.8 μM and 150-fold as control medium (no mentioned cytokinins) after 45 days on M3 medium. About 44% of the mature embryos were converted into plantlets with poor root system after subculture on M4 medium. Root further development of regenerated plantlets was promoted by addition of activated charcoal (AC) to MS basal medium.     

Transformation of <Emphasis Type="Italic">Platymonas</Emphasis> (<Emphasis Type="Italic">Tetraselmis</Emphasis>) <Emphasis Type="Italic">subcordiformis</Emphasis> (Prasinophyceae,Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25°C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10⁷ cell ml⁻¹. The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10⁻⁵, and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.     

Cell walls as reservoirs of potassium ions for reversible volume changes of pulvinar motor cells during rhythmic leaf movements

The laminar pulvinus of primary leaves of Phaseolus coccineus L. was investigated with respect to the total K⁺ content, the apoplastic K⁺ content, and the water potential of extensor and flexor sections in relation to the leaf positions in a circadian leaf-movement cycle, as well as the cation-exchange properties of isolated extensor- and flexor-cell walls. Turgid tissue showed a high total but low apoplastic K⁺ content, shrunken tissue a low total but high apoplastic K⁺ content. Thus, part of the K⁺ transported into and out of the swelling or shrinking protoplasts is shuttled between the protoplasts and the surrounding walls, another part between different regions of the pulvinus. The K⁺ fraction shuttled between protoplasts and walls was found to be 30–40% of the total transported K⁺ fraction. Furthermore, 15–20% of the total K⁺ content of the tissue is located in the apoplast when the apoplastic reservoir is filled, 5–10% when the apoplastic reservoir is depleted. The ion-exchange properties of walls of extensor and flexor cells appear identical in situ and in isolated preparations. The walls behave as cation exchangers of hhe weak-acid type with a strong dependence of the activity of fixed negative charges as well as of the K⁺-storing capacity on pH and [K⁺] of the equilibration solution. The high apoplastic K⁺ contents of freshly cut tissues reflect the cation-storing capacity of the isolated walls. We suggest that K⁺ ions of the Donnan free space are used for the reversible volume changes (mediating the leaf movement) mainly by an electrogenic proton pump which changes the pH and-or the [K⁺] in the water free space of the apoplast.Abbreviations and symbols DFS Donnan free space - DW dry weight - pK negative logarithm of the equilibrium constant K of the acidic group - WFS water free space - water potential; Indices - cw cell wall - t tissue     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

Production of protoplasts from Fusarium scirpi by lytic enzymes from Streptomyces tsusimaensis

Summary The ability of serveral strains of Streptomyces to degrade cell walls from Fusarium scirpi was tested by plating them on agar containing a cell wall preparation derived from the fungus. In this assay, S. tsusimaensis was most effective in producing a clear zone of lysis during growth on the opaque medium. This Streptomyce strain was subsequently grown in liquid culture containing cell walls as the sole carbon source and the exoenzymes were isolated from the culture broth. The enzyme preparation produces a clear zone of lysis when filled into wells in the cell wall agar and was used to prepare protoplasts from F. scirpi. The protoplast yield was 1x10⁹ protoplasts/ml of enzyme solution from 35 mg dry weight of Fusarium mycelium. Protoplasts could be regenerated at a frequency of up to 80%.